To try to develop highly sensitive, non-invasive diagnostic methods, we have been evaluating polymerase chain reaction (PCR) using primers based on the major surface glycoprotein (MSG) genes of human Pneumocystis. PCR using primers based on this gene is potentially a highly sensitive method since this is a multicopy gene (estimated at approximately equal to 50-100 copies/genome). We have been evaluating the diagnostic potential using a conserved region of the gene family. Our studies have shown that the sensitivity of MSG-based primers is greater than that of previously utilized primers. We are currently evaluating these primers prospectively in collaboration with the Microbiology Department and investigators at SFGH using respiratory samples including oral washes as the clinical specimen. As part of our collaboration with investigators at UCSF and Makerere University, we have been able to demonstrate that Pneumocystis is uncommon in HIV-infected patients in Uganda, which may be in part a result of the widespread use of trimethoprim-sulfamethoxazole prophylaxis in Uganda. We have developed and evaluated two typing techniques for human Pneumocystis. The first uses tandem repeats that occur in an intron of the MSG gene. By sequencing multiple MSG genes in a number of human Pneumocystis isolates, we have been able to demonstrate that recombination occurs in human Pneumocystis. These results led to studies that demonstrated that the MSG repertoire in human Pneumocystis is very diverse, while the MSG repertoire in rat and mouse Pneumocystis is identical or very limited among different isolates. Based on these studies, we have developed a restriction fragment length polymorphism (RFLP) typing assay for human Pneumocystis, and have been able to demonstrate substantial diversity among human isolates. Using RFLP analysis, we have been able to demonstrates that all the isolates from an outbreak of PCP in renal transplant patients in Germany appear to be the same strain, demonstrating that recent infection is important, and that either host to host transmission has occurred or that all individuals ere infected from a common source. We subsequently showed that the same isolate was responsible for an outbreak of PCP in renal transplant patients in Zurich, Switzerland, raising the possibility that this strain may be particularly virulent in renal transplant patients. To address this further, we examined samples from an outbreak of PCP in Japan. We found that while a single organism was responsible for the outbreak there as well, it was different from the European strain based on RFLP analysis. We have recently received samples from outbreaks in Denmark and in France for characterization by RFLP, and are in the process of examining these samples by RFLP as well as by multi-locus sequence typing (MLST). MLST is the most commonly used method for typing Pneumocystis in other laboratories, and this will allow us to compare the results of these two typing methods. These studies should provide improved diagnostic methods for PCP, and help to better understand the epidemiology of Pneumocystis infection.